|Title:||Application for Senior Investigator Authorization|
|Format:||Adobe Acrobat (PDF)1||Microsoft Word/Excel|
|Size:||410 KBytes||Not Available|
Instructions and Examples:
* ITEMS 1 – 6 are self explanatory.
* ITEMS 7 – 16 are explained below with step-by-step instructions and examples.
7. “Radioisotope(s) including element and mass number for which authorization is requested.”
|b) Carbon||14 C|
8. “Chemical and/or physical form of each radioisotope requested in item 7.”
9. “The maximum number of millicuries of each radioisotope requested in item 7.” (The maximum number of millicuries you will have stored in your lab at any one time through the year). This limit applies to the radioisotope only; it is not a maximum limit for each chemical form requested.
|7.a) Leucine in liquid|
|7.b) Succinate in liquid|
10. “Provide experimental protocols for each radioisotope requested in item 7. The protocol must include a mathematical justification of each requested possession limit:
|7.a) 1 mCi|
|7.b) 1 mCi|
We are following the uptake and assimilation of 14C-succinate and 3H-leucine by bacteria under a variety of conditions. These experiments require eight 50 ml cultures containing 0.1 uCi per ml 3H-leucine and 0.1 uCi per ml 14C-succinate. The cell suspensions are incubated 1-6 hours with sampling at regular intervals (every 10, 15, or 30 minutes, depending on the experiment). The samples are used to follow a number of parameters: turbidity, extracellular enzyme activity, residual radioactivity of growth medium, assimulation of label into cell material (protein), and cell dry weight. During the incubation period we expect 10-70% of the leucine and 25-90% of the succinate to be taken up by the bacteria. The efficiency of uptake and incorporation of label into the protein varies widely with culture conditions (ph, temperature, presence, or absence of antibiotics, population density, concentration of nutrients, etc). We desire a protein sample to have a counting level of about 25,000 cpm per ml of culture suspension. The counting efficiency of our Beckman liquid scintillation system is 92% for 14C and 60% for 3H. Experiments of this kind are conducted at a frequency of three per week with each experiment being repeated at least once.
JUSTIFICATIONS OF AMOUNT OF MATERIAL USED PER CULTURE:
Since we need 25,000 cpm/ml counting levels this requires the addition of 0.1 uCi/ml of 3H labeled leucine,
------------------------------------------------------- = 0.1 uCi/ml
(0.1)(0.6 cpm/dpm)(2.2 x 10-6 dpm/uCi)
and .05 uCi/ml of 14C labeled succinate,
--------------------------------------------------------- = 0.05 uCi/ml
(0.25)(.92 cpm/dpm)(2.2 x 10-6 dpm/uCi)
to each culture. However, since we desire to add equal amounts of labeled compound to the culture and the 3H labeled leucine is dominant, 0.1 uCi/ml of 14C-succinate will be used.
Each culture has a volume of 50 ml. Therefore we will use (0.1 uCi/ml x 50 ml/culture x 8 cultures/experiment) 40 uCi of the 3H and 14C labeled compounds per experiment.
JUSTIFICATION OF POSSESSION LIMIT:
We conduct these experiments at least 3 times per week consuming (40 uCi/experiment x 3 experiments per week) 120 uCi/week of the 3H and 14C. We would like to have enough label in the lab to last about 2 months; therefore 1 mCi of each label is required.
11. “Provide one copy of your Curriculum Vitae”.
12. & 13. Document your training required by law by filling out the "Radiological Training Record" (EHSF0003) form.
14. “Describe laboratory facilities for handling radioactive material.” For example: fume hoods, storage containers, shielding, etc.
Our work with 3H and 14C will be carried out in laboratory #222 Life Sciences building.
Extractions of aliquots from stock bottles will be done in fume hoods A and B on attached sketch. Stock bottles will be placed in a drip tray lined with absorbent paper to reduce the consequences of accidental spills of radioactive materials. These aliquots will be added to the culture in the fume hood. This step of our procedure will be carried out by individuals wearing protective poly gloves.
Labeled cultures will be incubated at 30*C in the oven marked “C” on the sketch.
Assay samples will be prepared on the benches marked D, E, and F on the sketch. No other lab benches will be used for radioactive work.
All radioactive waste will be discarded in containers provided for this purpose by the Radiation Safety Division.
Storage of radioactive materials will be in the freezer section of refrigerator G on the sketch. Shielding of the storage location and working areas is not necessary due to the low energy radiation emissions of 3H and 14C.
15. “Attach explanatory sketch of the laboratory facility.”
16. “Give a detailed description of the method to be used for the disposing of radioactive wastes and estimate the type and amount of activity involved [refer to EH&S waste disposal poster guide].”
Waste disposal explained in item 14 above. Estimate 1 mCi of 3H and 1 mCi of 14C every 2 months.